1. Field of the Invention
The present invention relates to a multilayer analytical element for assaying fructosamine in a body fluid, in particular, serum.
2. Description of the Related Art
For the measurement of fructosamine in serum, Johnson and Baker proposed a method comprising measuring the reducing power of fructosamine with Nitrotetrazolium Blue (NTB) under alkaline conditions (see Clinica Chimica Acta, 127 (1982) 87-95, Japanese Patent Publication No. 13062/ 1989, EP-A-085 263 and U.S. Pat. Nos. 4,642,295 and 4,645,742). In this method, the serum is preincubated for 10 minutes to remove the influence of reducing substances in the serum other than fructosamine by reacting such reducing substances with NTB to decompose them, and then the amount of NTB consumed in the following 5 minute period (namely from 10 minutes to 15 minutes) is measured since the amount of NTB which is consumed in this 5 minute period is proportional to the amount of fructosamine in the serum.
Japanese Patent Kokai Publication No. 182567/1988 and EP-A-215 170 disclose an end point assay in which interfering substances present in a sample are removed by treating them with a strong base, an oxidizing agent, or an enzyme, or by desalting, the pH of the sample is adjusted to a range between 10 and 14, a color reagent (a tetrazolium salt) is added, and the developed color is measured.
Japanese Patent Kokai Publication No. 15168/1988 and EP-A-250 991 disclose a method comprising pretreating a sample at neutral pH to remove reducing substances and/or turbid materials, adjusting the pH to a range between 10 and 12, and adding a color reagent.
Japanese Patent Kokai Publication No. 304999/1988 and EP-A-291 060 disclose a method comprising adding a buffer solution having a pH of 9 to 12, a color reagent, uricase and at least one detergent (surfactant) and, after 5 minutes at the earliest, chemically kinetically measuring the change in absorbance with time in the temperature range between 20.degree. C. and 40.degree. C.
The method of Johnson and Baker is a so-called wet chemistry method, and adjusts the temperature at 37.degree. C. under alkaline conditions and then measures the change of absorbance in a time range between 10 minutes and 15 minutes.
The method of Japanese Patent Kokai Publication No. 182567/1988 and EP-A-215 170 is a two-step method, which is troublesome and time-consuming.
In the method of Japanese Patent Kokai Publication No. 304999/1988 and EP-A-291 060, the preincubation requires at least 5 minutes before the reducing substances are removed and turbidity is clarified.
In addition, all the above prior art methods involve wet chemistry, and measure the change of absorbance using a reaction cell.
In general, formazan, which is generated by reduction of a tetrazolium salt to be used in a color developing reaction with fructosamine, firmly sticks to the reaction cell or the flow cell and increases the background, which is a source of error. When fructosamine is measured with an automatic analyzer which is widely used in many facilities, generated formazan deposits in the reaction tube, the transportation tube, or the flow cell contaminate the automatic analyzer, so that other components cannot be measured. Since it is difficult to remove deposited formazan, various devices or parts should be immersed in a strong acid or a surfactant overnight.
A reagent for measuring fructosamine is a strong alkaline reagent, for example, having a pH of 10 or greater, and its waste liquid is strongly alkaline and pollutes the environment.
In contrast to the wet chemistry method, a so-called dry chemistry method is known. In the dry chemistry method, since one drop (several .mu.l) of the sample is used for the reaction, the sample is dried when the reaction continues more than 10 minutes and accuracy in measurement deteriorates. When the measuring time is shortened to 10 minutes or less, the measurement is influenced by the reducing substances in the body fluid, so that accurate data are not obtained.